The present invention relates to improved retroviral vectors for gene therapy. Particularly, this invention relates to safe and efficient retroviral expression vectors, where all of the retroviral genes, i.e. gag, env and pol, are deleted completely; where a heterologous intron, splicing acceptor, and/or non-coding sequence are/is inserted into the upstream position of cloning site for a foreign gene; where a heterologous internal promoter or an internal ribosomal entry site (hereinafter, referred to as xe2x80x9cIRESxe2x80x9d) is inserted into the downstream position of the cloning site; and where the full-length U3 sequence of 5xe2x80x2 LTR (Long Terminal Repeat) or a strong heterologous promoter controls the expression of the foreign gene.
Retroviral vectors have been used for gene therapy more frequently than any other vector, being employed in more than 50% of the approved clinical protocols worldwide (Wileyxe2x80x94Journal of Gene Medicine Website. Although Murine Leukemia Virus (hereinafter, referred to as xe2x80x9cMLVxe2x80x9d)-based vectors are used dominantly, there are still many problems with the retroviral vectors in clinical use. The most serious problem is their safety; retroviral vector is one of the viral vector and thus may be converted in cells into replication-competent retrovirus (hereinafter, referred to as xe2x80x9cRCRxe2x80x9d) Above all, RCR production through homologous recombination has been a matter of grave concern.
All available retroviral vectors contain significantly long viral coding sequences. Since these sequences are also present in the genome of packaging cells from which the packaged vectors are released, it has bees suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of RCR, which was reported by Miller et al. (Miller et al., Human Gene Ther., 1: 5, 1990).
Two types of MLV-based retroviral vectors, LN series vectors and MFG vector, have been most frequently used for gene therapy (Miller and Roseman, Biotechniques, 7:980-990, 1989; Dranoff et al., Proc. Nat""l. Acad. Sci. USA 90: 3539-3543, 1993). While the expression of a foreign gene in LN series vectors is controlled by a heterologous internal promoter or by LTR, transcription level in MFG vector is under the control of LTR, and the foreign gene is expressed as a form of either genomic RNA or spliced subgenomic RNA. The LN series vectors, that are often considered the first generation retroviral vectors, contain the 420-bp gag coding sequence. Although this region has been thought to play an important role in viral packaging, it has been disclosed that gag region can be deleted without any significant effect on the viral packaging and titer under some conditions (Kim et al., J. Virol., 71: 994-1004, 1998).
Compared with LN series vectors, MFG vector is known to drive more stable and higher levels of gene expression, and produce higher viral titers in most of human- or mouse-derived cell lines (Byun et al., Gene Ther., 3: 780-788, 1996). However, MFG vector contains even more viral coding sequences, 420-bp for gag, 377-bp for pol, and 99-bp for env, raising the possibility of even higher frequency of producing RCR than the LN series vectors.
To overcome these disadvantages associated with conventional retroviral vectors, we, the inventors of this invention, constructed a retroviral vector (KOREA PATENT APPLICATION NO: 97-48095), which had several features as follows:
transcripts of the cloned gene was effectively spliced, producing higher expression levels of the gene,
gag and env sequences were completely deleted without a loss of viral titer,
IRES was used for the simultaneous expression of two or more genes in a vector,
multicloning site was inserted into the vector to facilitate the cloning of foreign gene
Since gag and env sequences were deleted from the retroviral vectors, the safety of the vectors was increased when compared with those of other retroviral vectors. However, this vector still contains the 377-bp pol coding sequence that harbors the splicing acceptor sequence as well as its downstream sequence containing the 284-bp leader (transcribed but untranslated) sequence for env, because the deletion of pol sequence would lead to abnormal or inefficient splicing. Since the 377-bp for pol sequence is also present in the genome of packaging lines, the possibility of homologous recombination resulting in RCR production still remains in the vector.
To develop novel retroviral vectors with elevated efficiency of gene expression as well as enhanced safety, we have constructed the retroviral vectors that do not contain ;any viral coding sequence. This invention is performed by constructing retroviral vectors, where MLV-derived pol gene is completely deleted, excluding the possibility of RCR production through homologous recombination in packaging cell line; where a heterologous intron, splicing acceptor, and/or non-coding sequence are/is inserted into the upstream position of cloning site for a foreign gene, maximizing the efficiency of gene expression; where either 5xe2x80x2 LTR or human cytomegalovirus (hereinafter, referred to as xe2x80x9cHCMVxe2x80x9d) major immediate early promoter (MIEP: the promoter of ie1 gene) is employed as cis-element for the regulation of foreign gene expression; and where either a heterologous internal promoter or an IRES is inserted in the the downstream position of the cloning site, allowing the simultaneous expression of two or more foreign genes.
It is an object of this invention to provide safe retroviral vectors where foreign gene(s) is/are expressed in higher levels and thus can be efficiently used for gene therapy.
In accordance with the present invention, the foregoing object is readily obtained.
This invention provides MLV-based retroviral vectors wherein the MLV-coding gag, env and pol sequences are completely deleted.
In one aspect, the MLV-based retroviral vectors of this invention may contain a heterologous intron and/or a heterologous splicing acceptor which are/is inserted into the upstream position of cloning site for a foreign gene.
In another aspect, the MLV-based retroviral vectors of this invention may contain a heterologous non-coding sequence inserted into the upstream position of cloning site for a foreign gene.
In further aspect, the full-length U3 sequence (xe2x88x92419 to xe2x88x921 bp) of MLV 5xe2x80x2 LTR may be replaced with a heterologous promoter in the MLV-based retroviral vectors of this invention.
In still further aspect, the MLV-based retroviral vectors of this invention may contain a heterologous promoter in the downstream position of cloning site for a foreign gene.
This invention also provides the E. coli strains transformed with the MLV-based retroviral vectors of this invention.
Further features of the present invention will appear hereinafter.